This invention relates to epi-fluorescent microscopy, and more particularly to the rapid selection of filters for measuring fluorescence at different wavelengths or Stokes shifts.
Current fluorescent microscope designs employ an incident light or epi-fluorescent design where a dichroic beam splitter (or chromatic mirror) mounted in a filter cube at a 45 degree angle to the excitation light path, is used to reflect shorter excitation wavelengths of light onto the specimen while passing longer emission wavelengths to the eyepieces or camera (FIG. 2).
Many fluorescence applications require two or more fluorescent labels to be present in the specimen. Each label has its own excitation and emission spectra, and thus requires different excitation and emission filters, as well as a different dichroic beamsplitter.
To date two approaches to using multiple fluorophores have been employed:                1) Use dichroic beamsplitters that have multiple cutoff wavelengths. Thus a single dichroic can be used with multiple fluorophores. However because of bandwidth restrictions, total light throughput is reduced, thus creating longer exposure times when working with a camera. Longer exposure times translate into longer acquisition duty cycles which is problematic in paradigms into longer acquisition duty cycles which is problematic in paradigms requiring repetitive high-speed data acquisition (screening applications and applications using living cells).        2) Motorized filter cube changers. Several commercially available microscopes employ motorized filter cube changes. These allow the use of single dichroics for each fluorophore. However switching time is slow (1-2 seconds) which creates problems when using multiple fluorophores in paradigms requiring repetitive high-speed data acquisition (screening applications and applications using living cells).        